Prokaryotic Expression Of Brucella Bp26 Protein And Establishment Of IELISA Based On The Recombinant Bp26 Protein

Brucellosis is a zoonotic disease caused by Brucella,which not only causes abortions of infected animals,inflammation of the genital organs,infertility and local lesions of various tissues,but also causes human patients debilitate and multiple system inflammation.Brucellosis seriously threaten the animal husbandry and people’s life and health.As livestock,especially sheep,are the major source of brucellosis,the detection and control of brucellosis in livestock is particularly critical.However,most brucellosis detection methods used currently have low sensitivity,time-consuming and laborious,and can not distinguish between natural infection and vaccine immunity.Therefore,it is very necessary to establish a simple,convenient,sensitive and specific detection method.ELISA detection method has the advantages of time-saving and labor-saving,high sensitivity,and has been well applied in the detection of many diseases,laying a solid foundation for the application of this method in brucellosis detection.In addition,some studies have shown that the bp26 gene is highly conserved but differentially expressed on wild-type Brucella and Brucella vaccine strains,and thus the bp26 gene has the potential to be used as a test antigen for distinguishing between natural infection and vaccine immunity.In this study,Brucella periplasmic protein bp26 was used as an antigen to establish a ELISA method for detecting brucellosis in goat.Firstly,specific PCR primers were designed according to the bp26 gene sequence published on GenBank.The genomic DNA of Brucella spp.S2 strain as the template to amplify bp26 gene.Then bp26 gene was ligated into pGEX-4T-1 vector to construct pGEX-bp26 recombinant expression vector,and the recombinant expression vector was transformed into BL21(DE3)competent cells,and then expresse and purify the GST-bp26 recombinant protein.Finally,the purified recombinant protein was used as coating antigen,and then the optimal antigen coating concentration,the optimal dilution of primary antibody,the optimal coating time,the optimal blocking solution and blocking time,the best primary antibody and secondary antibody incubation time,the best color development time were identified.The results are as follows:(1)The prokaryotic expression vector of Brucella bp26 gene was constructed,and the recombinant protein of GST-bp26 was induced and purified.(2)The method of detecting brucellosis in goat by ELISA was established by using the purified recombinant protein.The reaction conditions were as follows:the concentration of antigen coating was 3.2 μg/mL,the best dilution of primary antibody was 1/10,the best coating time was 37℃ for 1 h,the best blocking solution was 5%skimmed milk,the best blocking time was 37℃ for 30 min,the best first antibody incubation time was 37℃ for 1 h,the best second antibody incubation time was 37℃ for 1 h,and the best color time is 37℃for 30 min.The iELISA assay established in this study has good specificity and sensitivity,and has good conformance with the RBT,which can meet the clinical testing needs of brucellosis and has good clinical value.